Abstract 1220 Characterization of Xenobiotic response element (XRE) family transcription factor and its role in the regulation of dsz operon

Poor understanding of new antimicrobial mechanisms of action can impede compound prioritization for further development.Chemical genetics, which links bioactive compounds with their cellular targets, offers a streamlined approach to antibiotic discovery.Here, we generated a de novo chemicalgenetic interaction dataset in the multi-resistant bacterium Burkholderia cenocepacia with a broad coverage of chemicals.Utilizing a CRISPR interference-mediated pooled library of essential genes (CIMPLE) followed by CRISPRi-seq (CIMPLEseq), we screened 5,000 compounds, generating over 3.2 million chemical-genetic interactions.Our primary screen yielded 32-fold more hits than whole-cell, high-throughput approaches against wild-type bacteria.With a Z-score threshold of -5, approximately 28% of compounds depleted at least one knockdown mutant from the CIMPLE pool compared to the DMSO control.Notably, we observed MOAbased clustering of compounds independent of the compound structures.Our analysis identified compounds targeting essential cellular processes, including DNA gyrase, folate biosynthesis, ribosomal proteins, and RNA polymerase.We also identified inhibitors of the tsaE/amiC operon, an unexplored antibiotic target involved in cell wall degradation at the divisional septum.Finally, a secondary screening assay was developed employing selective media and fluorescent protein tags to confirm competitive fitness during co-culture growth.The assay identified enhanced sensitivity in essential gene knockdown mutants of known antibiotic targets, revealing its potential for confirmatory testing of chemical-genetic profiles.These findings demonstrate the sensitivity and specificity of our approach in elucidating chemical-genetic interactions of uncharacterized bacterial growth inhibitors from new classes.